Nuisance compounds in cellular assays.

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity ("nuisance compounds") are routinely encountered in cellular assays, including phenotypic and high-content screening assays. Much is known regarding compound-dependent assay interferences in cell-free assays. However, despite the essential role of cellular assays in chemical biology and drug discovery, there is considerably less known about nuisance compounds in more complex cell-based assays. In our view, a major obstacle to realizing the full potential of chemical biology will not just be difficult-to-drug targets or even the sheer number of targets, but rather nuisance compounds, due to their ability to waste significant resources and erode scientific trust. In this review, we summarize our collective academic, government, and industry experiences regarding cellular nuisance compounds. We describe assay design strategies to mitigate the impact of nuisance compounds and suggest best practices to efficiently address these compounds in complex biological settings.

Second-Harmonic Generation (SHG) for Conformational Measurements: Assay Development, Optimization, and Screening.

Second-harmonic generation (SHG) has recently emerged as a biophysical tool for conformational sensing of a target biomolecule upon binding to ligands such as small molecules, fragments, proteins, peptides, and oligonucleotides. To date, SHG has been used to measure conformational changes of targets such as soluble proteins, protein complexes, intrinsically disordered proteins, peripheral and integral membrane proteins, peptides, and oligonucleotides upon binding of ligands over a wide range of affinities. In this chapter, we will provide a technology overview, detailed protocols for optimizing assays and screening, practical considerations, and an example case study to guide the reader in developing robust and informative measurements using the Biodesy Delta SHG platform.

Chemical Proteomic Characterization of a Covalent KRASG12C Inhibitor.

The KRASG12C protein product is an attractive, yet challenging, target for small molecule inhibition. One option for therapeutic intervention is to design small molecule ligands capable of binding to and inactivating KRASG12C via formation of a covalent bond to the sulfhydryl group of cysteine 12. In order to better understand the cellular off-target interactions of Compound 1, a covalent KRASG12C inhibitor, we have completed a series of complementary chemical proteomics experiments in H358 cells. A new thiol reactive probe (TRP) was designed and used to construct a cellular target occupancy assay for KRASG12C. In addition, the thiol reactive probes allowed us to profile potential off-target interactions of Compound 1 with over 3200 cysteine residues. In order to complement the TRP data we designed Compound 2, an alkyne containing version of Compound 1, to serve as bait in competitive chemical proteomics experiments. Herein, we describe and compare data from both the TRP and the click chemistry probe pull down experiments.

A Perspective on the Kinetics of Covalent and Irreversible Inhibition.

The clinical and commercial success of covalent drugs has prompted a renewed and more deliberate pursuit of covalent and irreversible mechanisms within drug discovery. A covalent mechanism can produce potent inhibition in a biochemical, cellular, or in vivo setting. In many cases, teams choose to focus on the consequences of the covalent event, defined by an IC50 value. In a biochemical assay, the IC50 may simply reflect the target protein concentration in the assay. What has received less attention is the importance of the rate of covalent modification, defined by kinact/KI. The kinact/KI is a rate constant describing the efficiency of covalent bond formation resulting from the potency (KI) of the first reversible binding event and the maximum potential rate (kinact) of inactivation. In this perspective, it is proposed that the kinact/KI should be employed as a critical parameter to identify covalent inhibitors, interpret structure-activity relationships (SARs), translate activity from biochemical assays to the cell, and more accurately define selectivity. It is also proposed that a physiologically relevant kinact/KI and an (unbound) AUC generated from a pharmacokinetic profile reflecting direct exposure of the inhibitor to the target protein are two critical determinants of in vivo covalent occupancy. A simple equation is presented to define this relationship and improve the interpretation of covalent and irreversible kinetics.

The Use of Nucleosome Substrates Improves Binding of SAM Analogs to SETD8.

SETD8 is the methyltransferase responsible for monomethylation of lysine at position 20 of the N-terminus of histone H4 (H4K20). This activity has been implicated in both DNA damage and cell cycle progression. Existing biochemical assays have utilized truncated enzymes containing the SET domain of SETD8 and peptide substrates. In this report, we present the development of a mechanistically balanced biochemical assay using full-length SETD8 and a recombinant nucleosome substrate. This improves the binding of SAM, SAH, and sinefungin by up to 10,000-fold. A small collection of inhibitors structurally related to SAM were screened and 40 compounds were identified that only inhibit SETD8 when a nucleosome substrate is used.

Beyond PAINs: Chemotype Sensitivity of Protein Methyltransferases in Screens.

Screening of the relatively new target class, the lysine and arginine methyltransferases (MTases), presents unique challenges in the identification and confirmation of active chemical matter. Examination of high throughput screening data generated using Scintillation Proximity Assay (SPA) format for a number of protein MTase targets reveals sensitivity to both the known pan assay interference compounds (PAINS) and also other scaffolds not currently precedented as assay interferers. We find that, in general, true actives show significant selectivity within the MTase family. With the exception of slight modifications of SAM-like compounds, scaffolds that are observed frequently in multiple MTase assays should be viewed with caution and should be carefully validated before following up.

The identification of beta-hydroxy carboxylic acids as selective MMP-12 inhibitors.

A new class of selective MMP-12 inhibitors have been identified via high throughput screening. Crystallization with MMP-12 confirmed the mode of binding and allowed initial optimization to be carried out using classical structure based design.

Thiol-based angiotensin-converting enzyme 2 inhibitors: P1 modifications for the exploration of the S1 subsite.

Screening of a metalloprotease library led to the identification of a thiol-based dual ACE/NEP inhibitor as a potent ACE2 inhibitor. Modifications of the P(1) benzyl moiety led to improvements in ACE2 potency as well as to increased selectivity versus ACE and NEP.